TABLE 1.
Systematic comparison of barcoding strategies used in the category of molecular barcodes.
| Approach | Type | Strategy | Combinations of different pairs of barcoding | PCR required for barcoding | Target SARS-CoV-2 gene(s)/protein(s) | Commercial kit used for barcoding | Sample type | Platform/Experimental pipeline | Required software a | Functions of barcodes | Major contribution of the technology | Ref. |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Primer-associated approach | Sequence-based barcodes | SQK-RBK004: transposase carrying barcodes to the site of the cleavage | - | - | Whole genome | Oxford Nanopore Rapid Barcoding kit (SQK-RBK004) | SARS-CoV-2 patient samples (nasopharyngeal swab) | Oxford Nanopore | Guppy version 3.6.0; ARTIC Network bioinformatics protocol | Multiplex samples | Propose a method to sequence the whole genome of SARS-CoV-2 in a rapid and cost-efficient manner | Freed et al. (2020) |
| Protein-protein interaction approach | Sequence-based barcodes | A pair of DNA barcodes is installed on the SARS-CoV-2 spike protein S1 subunit | - | - | S | n/a | 57 blood specimens | Hamilton Microlab ADAP STAR automated liquid-handling platform | PRISM v8.1.1; XLSTAT software 2019.1 | Quantify protein-antibody interaction | Qualitative detection of total antibodies against S1 subunit of the spike protein | Karp et al. (2020) |
| CRISPR-associated approach | Sequence-based barcodes | Unique sgRNA sequences that serve as unique barcodes are encoded on the same plasmid used for making the dCas9-fusion library | - | - | S, N | n/a | 6 COVID-19 convalescent samples | DNA microarray platform (GenePix 4300A microarray scanner) | Microarray data processing scripts (GitHub); GenePix Pro 7 | Multiplex samples | Characterization of (polyclonal) antibody-epitope binding | Barber et al. (2021) |
| Primer-associated approach | Sequence-based barcodes | Tagmentation performed by bead-linked transposes and PCR amplification | - | + | ORF1ab, S, E, M, N | IDT for Illumina Nextera UD Indexes Set A, B, C, D (384 indexes, 384 samples) | 752 patient sample (nasopharyngeal swab, oropharyngeal swab, and nasal swab) | Illumina NovaSeq 6000 | Illumina DRAGEN COVIDSeq Test Pipeline | Multiplex samples | Population diagnostics (1536 sequencing libraries) and a confirmatory test; variants analysis | Bhoyar et al. (2021) |
| Primer-associated approach | Sequence-based barcodes | Barcodes embedded in the RT-PCR primers | + | + | S, N | n/a | Extraction-free lysates from mid-nasal swabs and saliva | Illumina MiSeq, MiniSeq and the Illumina NextSeq 550 systems | R package swabseqr | Multiplex samples | Population diagnostics (80,000 samples) | Bloom et al. (2021) |
| Primer-associated approach | Sequence-based barcodes | Barcodes embedded in the RT primers | + (barcode and UMI) | + | N | n/a | Hospitalized patient samples (5 s of exhaled breath was collected in the Bubbler) | Bubbler breathalyzer | Bowtie software version 2.2.4 | Multiplex samples | Direct detection of SARS-CoV-2 from exhaled breath | Duan et al. (2021) |
| Primer-associated approach | Sequence-based barcodes | SISPA† barcoding primers are given at the step of RT; Ligation of barcodes (Oxford Nanopore sample indexes) to both ends of DNA sequencing amplicons | + (SISPA dual barcodes + Oxford Nanopore barcodes) | + | Whole genome | Native barcoding expansion 96 kit (EXP-NBD196) | 43 clinical specimens (oropharyngeal swab and nasopharyngeal swab) | Oxford Nanopore | Guppy (Version 5.0.7, Oxford Nanopore Technologies); BugSeq (version 1.1, database version: RefSeq on 28 Jan 2021) | SISPA barcoding primers enable to detect and assemble genomes of SARS-CoV-2; Oxford Nanopore barcoding used for multiplexing samples | Variants analysis | Gauthier et al. (2021) |
| Primer-associated approach | Sequence-based barcodes | SQK-RBK004: transposase carrying barcodes to the site of the cleavage; no barcodes are used in the kit SQK-LSK109 | - | - | Whole genome | The Rapid Barcoding kit (SQK-RBK004) and the Ligation Sequencing Kit (SQK-LSK109) | Clinical samples | Oxford Nanopore | Guppy version 4.0.11 (community.nanoporetech.com) and the high accuracy version of the flip-flop algorithm | Multiplex samples | Comparison of two kits used for the whole SARS-CoV-2 genome sequencing | González-Recio et al. (2021) |
| Primer-associated approach | Sequence-based barcodes | LAMP barcodes embedded in the forward inner primer (FIP); PCR barcodes (indexes, i5 and i7) introduced at the PCR stage | + (LAMP-barcodes + PCR-barcodes) | + | E, N | n/a | SARS-CoV-2 swab samples | Illumina MiSeq or iSeq sequencer | LAMP-Seq Inspector so ware (http://manuscript.lamp-seq. org/Inspector.htm) | LAMP barcodes: specify each sample; PCR barcodes: multiplex samples | Population diagnostics (676 samples) | Ludwig et al. (2021) |
| Direct ligation approach | Sequence-based barcodes | Unique barcodes are generated by combining different A and B oligos | - | - | Whole genome | n/a | PBMCs from 18 SARS-CoV-2-infected patients | Ion Torrent PGM 314 or 316 chip (Life Technologies) | ‘Barracoda’ (https://services.healthtech.dtu.dk/service.php?Barracoda- 1.8) | Multiplex samples | Identification of immunogenic CD8+ T cell epitopes | Saini et al. (2021) |
| Primer-associated approach | Sequence-based barcodes | The first barcode is introduced in the primer for reverse transcription; the second barcode is given in the step of PCR amplification. This strategy is called “Concat-PCR” | + (the first- and the second barcodes) | + | E, S | Oxford Nanopore PCR Barcoding Expansion 1–96 kit (EXP-PBC096) | SARS-CoV-2 synthetic RNA | Oxford Nanopore | RETIVAD | Multiplex samples | Diagnostics (proof of concept); variants analysis | Stüder et al. (2021) |
| Staining approach | Color-based barcode | 106 Ramos B cells are resuspended in different concentrations of the cell proliferation tracer CytoTell blue | - | - | S, RBD | n/a | 12 COVID-19 patient samples | Color-based barcoded spike protein flow cytometric assay (BSFA) | n/a | Label and separate samples | Comparison of immune responses triggered by different variants of SARS-CoV-2 | Vesper et al. (2021) |
| Primer-associated approach | Sequence-based barcodes | Two unique barcodes embedded in primers at the stage of RT | + (the left and the right barcodes) | + | S | n/a | Commercial pooled human saliva from healthy individuals with spiked-in synthetic viral RNA | INSIGHT [isothermal NASBA (nucleic acid sequence–based amplification) sequencing–based high- throughput test]; Illumina MiSeq (PE 150bp) | FASTX_trimmer | Multiplex samples | Diagnostics (48 samples) | Wu et al. (2021) |
| CRISPR-associated approach | Sequence-based barcodes | Customized peptide libraries are designed to encode a unique 20 bp nucleic acid sequence used as the gRNA barcode | - | - | S | n/a | COVID-19 samples (convalescent, pre-vaccine and post-vaccine) | Cas9 display (CasPlay) system (GenePix 4300A microarray scanner); Illumina NextSeq 500 (single-end 150 bp) used to sequenced dCas9-fusion library | GenePix Pro 7; Cutadapt v2.5 Martin. (2011) and customized commend lines | Multiplex samples | Evaluation of vaccine-induced antibody reactivities from the SARS-CoV-2 proteome | Barber et al. (2022) |
| Primer-associated approach | Sequence-based barcodes | Barcodes embedded in the forward primer during the step of one-step-RT-PCR | - | + | ORF1, E, N1 | n/a | 960 Oro- and nasopharyngeal swabs collected from SARS-CoV-2 patients | Illumina Miseq sequencer (PE150 bp) | R package DNABarcodes created unique ten-base barcodes; FASTX-toolkit version 0.0.14 (http://hannonlab.cshl.edu/fastx_toolkit/) | Multiplex samples | Variants analysis | Cohen-Aharonov et al. (2022) |
| Primer-associated approach | Sequence-based barcodes | A barcode is introduced upstream of the ribosome-binding site in the recombined pDEST–MIPSA vector | - | + | n/a | n/a | 55 COVID-19 patient samples | MIPSA (Molecular Indexing of Proteins by Self-Assembly); Illumina MiSeq | The MIPSAlign package (https://github.com/jgunn123/MIPSAlign) | Individually label 12,680 human clonal open reading frames (mapped to 11,437 genes) | Identification of autoreactive antibodies in plasma samples | Credle et al. (2022) |
| Protein-protein interaction approach | Sequence-based barcodes | DNA barcodes are cross-linked on the S1 subunit protein and the ACE2 receptor | - | - | S1 | n/a | 146 COVID-19 serum samples | Split-Oligonucleotide Neighboring Inhibition Assay (SONIA) on the basis of real-time qPCR | n/a | Detect whether neutralizing antibodies block the binding between the S1 protein and the ACE2 receptor | Quantification of neutralizing antibodies binding on SARS-CoV-2 S protein subunit 1 | Danh et al. (2022) |
| Primer-associated approach | Sequence-based barcodes | Ligation of barcodes to both ends of DNA of interest after performing FFPE repair and end-prep | - | - | Whole genome and the spike region of interest (positions 23,468 to 23,821) | Native Barcode expansion kit (EXP-NBD196); Ligation sequencing kit (SQK-LSK109) | The hamster and pneumocyte samples | Oxford Nanopore | MinKNOW v4.3.7.; Guppy 5.0.12 | Multiplex samples | Variants analysis | Escalera et al. (2022) |
| Direct ligation approach | Sequence-based barcodes | A barcode is inserted in a string of the DNA sequence used to link to biotinylated spike ectodomain or spike-RBD | - | - | S (spike ectodomain and RBD) | Solulink Protein-Oligonucleotide Conjugation Kit (TriLink cat no. S-9011) | Mice models (BALB/c and C57BL/6J) | LIBRA-seq technology; single-cell RNA sequencing (10x Genomics User Guide, CG000186 Rev D) | CITE-seq-Count Mimitou et al. (2019) | Barcode on each B cell to indicate its antigen specificity | Identification of an antibody (SW186) that can neutralizes SARS-CoV-2 | Fang et al. (2022) |
| Primer-associated approach | Sequence-based barcodes | Spike-ins barcodes are prepared according to the PrimalSeq v.4.0 protocol Matteson et al. (2020) | + (Spike-ins barcodes + NEXTflex Dual-Indexed DNA Barcodes) | + | Whole genome | n/a | 49 SARS-CoV-2 patient samples | Illumina MiniSeq (PE150 bp) | Cutadapt v.2.10 (demultiplexing) Martin. (2011); iVar Grubaugh et al. (2019) | Spike-ins barcodes used to detect potential sample cross-contamination; indexed DNA barcodes: multiplexing samples | Reconstruction of SARS-CoV-2 transmission history | Gallego-García et al. (2022) |
| Antibody-staining approach | Sequence-based barcodes | Cells were incubated with mixtures of barcoded antibodies | - | - | n/a | TotalSeqTM-A Antibodies and Cell Hashing with 10x Single Cell 3′ Reagent Kit v3 3.1 Protocol (Biolegend) | Clinical PBMC samples from SARS-CoV-2 patients (277 TotalSeq-A antibodies) | TotalSeq antibodies in combination with single-cell RNA sequencing (Illumina NovaSeq S4 flow cell) | Cell Ranger 3.1.0 with default parameters ( https://github.com/10xGenomics/cellranger) | Multiplex samples | Benchmark different hashing methods for single-cell RNA-seq on clinical samples from SARS-CoV-2 patients | Mylka et al. (2022) |
| Primer-associated approach | Sequence-based barcodes | Barcodes embedded in primers to perform first-strafed cDNA | + (A well-specific barcode and a plate-specific barcode) | + | N | Illumina indexed barcoding kit (optional) | 4 SARS-CoV-2 patient samples | Reombinase mediated barcoding and amplification diagnostic tool (REMBRANDT) associated with Illumina sequencing platform (MiSeq) | The REMBRANDT pipeline ( https://github.com/MilesLab/Rembrandt_pipeline/) | Specify samples in each well and in each plat and multiplex samples for sequencing | Scalable diagnostic test (6 samples tested, 4 SARS-CoV-2-positive, 2 SARS-CoV-2-negative) | Palmieri et al. (2022) |
| Primer-associated approach | Sequence-based barcodes | Same design described in LAMP-Seq Ludwig et al. (2021) | + (LAMP-barcodes + PCR-barcodes) | + | N | n/a | Contrived saliva samples and 120 clinical nasopharyngeal swab samples | COV-ID pipeline: RT-LAMP combined with Illumina NextSeq or similar instrument | FASTX-toolkit utility fastq_quality_ lter ( http://hannonlab. cshl.edu/fastx_toolkit/); Cutadapt Martin. (2011) | See LAMP-seq Ludwig et al. (2021) | Scalable diagnostic test and an approach for the simultaneous detection of different pathogens, including SARS-CoV-2 in contrived saliva samples | Warneford-Thomson et al. (2022) |
| Primer-associated approach | Sequence-based barcodes | Patient barcodes (10 bp) introduced during the initiation of the RT step; plate barcodes given at the PCR step | + (Patient barcode and plate barcode) | + | Whole genome‡ | n/a | Synthetic RNA templates of SARS-CoV-2; 45 patient samples (nasopharyngeal swabs and saliva samples) | DeepSARS with the Illumina MiSeq system (2 × 81 and 1 × 150 cycle runs) | R package Rsubread | Multiplex samples | Scalable diagnostic test; variants analysis | Yermanos et al. (2022) |
| CRISPR-associated approach | Sequence-based barcodes | External barcodes (4 bp) added at the external region outside of sgRNA at its 3′end | - | - | S | n/a | n/a | High-throughput sequencing (not specified in this study) | eBAR-analyzer (https://github.com/wolfsonliu/FluorescenceSelection) | Execute a high multiplicity of infection (MOI) in generating the cell library for screening | Identify novel host factors required for SARS-CoV-2 entry | Zhu et al. (2022) |
a
Software indicated here are those used to proceed with raw sequencing reads.
†Sequence-independent single primer amplification (SISPA) (Reyes and Kim, 1991).
‡The whole genome sequence was obtained based on a multiple sequence alignment of short reads (110–140 bp) sequenced by Illumina MiSeq.